Method of producing and purifying soluble recombinant COQ5 protein and soluble recombinant COQ5 protein thereof

Abstract

The present invention relates to a method of producing and purifying soluble recombinant coenzyme Q5 homolog (COQ5) protein, which is expressed in soluble form by Escherichia coli ( E. coli ), under native conditions. The method is characterized by pre-treating bacterial lysate with low concentration of ionic detergent, such as sodium dodecyl sulfate (SDS), before purification; and the purifying method is performed under native condition without using urea to avoid the problems of requiring lengthy processes to remove urea in purified protein solution or re-aggregation and precipitation of protein after removal of urea.

What is claimed is:  
     
       1. A recombinant mitochondrial targeting signal (MTS)-truncated coenzyme Q5 homolog (COQ5) protein prepared by the process comprising the steps of:
 providing an  Escherichia coli  ( E. coli ) host cell transformed with an expression vector containing DNA fragments that encode the recombinant COQ5 protein, wherein said recombinant COQ5 protein contains a 6× histidine tag at the N-terminus; 
 inducing the expression of recombinant COQ5 protein in the  E. coli  host cell; 
 lysing the bacterial cells with a lysis buffer comprising lysozyme and deoxyribonuclease I (DNase I); 
 pre-treating the cell lysate with less than 0.2% of an ionic detergent; 
 centrifuging the cell lysate for removing insoluble proteins or debris and collecting supernatant containing the recombinant COQ5 protein; and 
 applying the supernatant to nickel-nitrilotriacetic acid (Ni-NTA) beads under native conditions that lack exposure to urea to obtain purified recombinant COQ5 protein; wherein the recombinant COQ5 protein remains soluble during purification and does not become aggregated or form precipitate after purification when protein solution is exchanged with buffer and the protein is concentrated. 
 
     
     
       2. The recombinant COQ5 protein of  claim 1 , wherein the recombinant COQ5 protein comprises the amino acid sequence of SEQ ID NO: 2 and is a histidine-tagged human COQ5 protein without a mitochondrial targeting signal (MTS). 
     
     
       3. The recombinant COQ5 protein of  claim 2 , which is used to prepare anti-COQ5 antibodies. 
     
     
       4. The recombinant COQ5 protein of  claim 3 , wherein the anti-COQ5 antibody is applied to detect endogenous human COQ5 protein in human cells or tissues.